Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MACl) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacteria1 artificial chromosome library. Sequence characterization showed that MACl has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MACl were unable t o form appressoria on an inductive surface and were unable t o penetrate susceptible rice leaves. m a c l -transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.

The PMK1 mitogen-activated protein kinase gene regulates appressorium formation and infectious hyphae growth in the rice blast fungus. To further characterize this mitogen-activated protein kinase pathway, we constructed a subtraction library enriched for genes regulated by PMK1 . Two genes identified in this library, GAS1 and GAS2 , encode small proteins that are homologous with gEgh16 of the powdery mildew fungus. Both were expressed specifically during appressorium formation in the wild-type strains, but neither was expressed in the pmk1 mutant. Mutants deleted in GAS1 and GAS2 had no defect in vegetative growth, conidiation, or appressoria formation, but they were reduced in appressorial penetration and lesion development. Interestingly, deletion of both GAS1 and GAS2 did not have an additive effect on appressorial penetration and lesion formation. The GAS1 -green fluorescent protein and GAS2 -green fluorescent protein fusion proteins were expressed only in appressoria and localized in the cytoplasm. These two genes may belong to a class of proteins specific for filamentous fungi and function as novel virulence factors in fungal pathogens.

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Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacterial artificial chromosome library. Sequence characterization showed that MAC1 has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate susceptible rice leaves. mac1- transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.

summary A system-wide approach was adopted to further elucidate mechanisms regulating disease outcome between rice and the fungal pathogen Magnaporthe grisea. First, a cDNA library was constructed from M. grisea infected rice at 48 h post-inoculation. The 5' end-sequencing of 619 randomly selected clones revealed 359 expressed sequence tags (ESTs) that had not previously been described. A total of 124 from 260 ESTs with high and moderate similarity scores, based on BlastX, were organized into categories according to their putative function. The largest category of sequences (21%) contained stress or defence response genes. Eleven per cent of identified ESTs were redundant. In a second approach, differential hybridization analysis of the cDNA library using high-density filters resulted in the identification of novel genes and previously characterized M. grisea genes, including several that had previously been implicated in the infection process. A survey of up-regulated cDNA clones revealed clone 29003, which corresponded to the rice peroxidase POX22.3. This gene is known to be expressed in rice upon infection with Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen. Importantly, this approach demonstrates the utility of gene discovery, through ESTs, for revealing novel genes in addition to those previously characterized as being potentially implicated in host-pathogen interactions.

The estrogen-related receptor (ERR) family of orphan nuclear receptor is composed of ERRα, ERRβ, and ERRγ, which are known to regulate various isoform-specific functions under normal and pathophysiological conditions. Here, we investigate the involvement of ERRs in the pathogenesis of osteoarthritis (OA) in mice. Among ERR family members, ERRγ is markedly upregulated in cartilage from human OA patients and various mouse models of OA. Adenovirus-mediated overexpression of ERRγ in mouse knee joint or transgenic expression of ERRγ in cartilage leads to OA. ERRγ overexpression in chondrocytes directly upregulates matrix metalloproteinase (MMP)-3 and MMP13, which are known to play crucial roles in cartilage destruction in OA. In contrast, genetic ablation of Esrrg or shRNA-mediated downregulation of Esrrg in joint tissues abrogates experimental OA in mice. Our results collectively indicate that ERRγ is a novel catabolic regulator of OA pathogenesis.

Porous capsules composed of hematite, silica, and hematite–silica composites are prepared by a templated synthesis method. Polyelectrolyte multilayer‐coated particles (PEMPs) are used to synthesize goethite nanocrystals and the resulting goethite‐nanocrystal‐embedded PEMPs (PEMP–goethite) are then used as templates to form porous capsules. The surface morphology and surface area of the porous capsules can be controlled by the size of the PEMP–goethite template, which is determined by the extent of growth of the goethite nanocrystals. By controlling the surface morphology and area, it is also possible to tune the sensitivity of the hematite capsules for use as gas‐sensing materials. This surfactant‐free approach is also used to synthesize silica and silica‐based composite capsules with a controllable porous shell thickness. This straightforward approach can also be extended to the synthesis of other porous capsules or particles with a controllable surface morphology.

A one-step, template-free synthetic method for preparing polymeric microcapsules with iron oxide (γ-Fe 2 O 3 ) magnetic nanoparticles (MPs) embedded in the polymer shell is reported. Using a simple emulsification of the multiphase mixture containing liquid prepolymer and MPs in chloroform solution, double emulsions comprising a chloroform core and MPs/polymer shell were spontaneously formed. After exposure to UV light, these double emulsions converted to microcapsules with a polymerized composite shell. The evolution from the double emulsions to the microcapsules was examined by confocal laser scanning microscopy. One unusual feature of these microcapsules is the ability to change shape reversibly by osmotic swelling of the water core upon repetitive drying and hydration. The microcapsules had an intrinsic superparamagnetic response due to the presence of the magnetic nanoparticles and could be moved and collected by external magnetic fields.